This proposal is designed to test the hypothesis that the release of acetylcholine (ACh) from motor nerves is modulated by a presynaptic cholinergic receptor (Pre-AChr). Recent evidence from my laboratory and others suggests that a Pre-AChR is present on motor nerve terminals and operates as a negative feedback system in monitoring and adjusting the amount of ACh released or releasable during stimulation. Antagonism of this Pre-AChR by agents such as d-tubocurarine or physostigmine results in an increase in the amount of ACh released on nerve stimulation as measured biochemically. Electrophysiological evidence for a Pre-AChR as obtained by intracellular recordings from the post-synaptic side of the neuromuscular junction has been equivocal since these agents directly effect the post-junctional AChR from which the electrical phenomena originate. The proposed study will address these disputations by directly measuring the release of endogenous ACh from a uniquely suited neuromuscular model system--the vascular perfused rat phrenic nerve-hemidiaphragm preparation--by means of a sensitive radioenzymatic assay for ACh. In order to characterize this putative Pre-AChR, the effects and interactions of specific cholinoceptor agonists and antagonists on the stimulated and unstimulated release and tissue concentrations of ACh will be investigated by a classical pharmacological approach of dose-response determinations. The results of these experiments will be interpreted with concomitant conventional intracellular recordings from the neuromuscular junction. In addition, the effects of precursor (choline) availability, and subsensitivity and supersensitivity of the Pre-AChR will be examined to determine if these conditions further modify the modulation of ACh release. Thus, the present proposal promises to increase our understanding of cholinergic biology with regard to the presynaptic modulation of acetylcholine release and has important implications for the development of therapeutic approaches which attempt to correct cholinergic dysfunction at its presynaptic origin.